Novel compositions, methods and kits for real time polymerase chain reaction (pcr)

ABSTRACT

The present disclosure is directed to compositions, methods and kits for amplifying target nucleic acids while reducing non-specific amplification and undesired amplification products using a dual hot start reaction mixture that comprise at least two different hot start mechanisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/927,572, filed Mar. 21, 2018, which is a divisional of U.S.application Ser. No. 13/918,768, filed Jun. 14, 2013, now U.S. Pat. No.9,951,378, which claims a priority benefit under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application No. 61/659,587, filed Jun. 14, 2012.The entire contents of the aforementioned applications are incorporatedby reference in their entireties herein.

FIELD

This disclosure generally relates to compositions, methods and kits foramplifying nucleic acids while reducing non-specific amplificationand/or undesired amplification products.

BACKGROUND

While the polymerase chain reaction (PCR) and related techniques arehighly useful for a variety of applications, the amplification ofnon-target nucleic acids due to undesired side-reactions can present asignificant problem. Such side reactions can occur as a result ofmis-priming of non-target nucleic acids and/or primer oligomerization,sometimes referred to as primer-dimer formation, and the subsequentamplification of these priming artifacts. This is especially true inapplications in which PCR is carried out using a mixture of nucleicacids with significant background nucleic acids while the target nucleicacid is present in low copy number (see, e.g., Chou et al., Nucl. AcidsRes. 20:1717 (1992)). The generation of non-specifically amplifiedproducts has been attributed at least in part to DNA polymerase activityat ambient temperature that extends non-specifically annealed primers(see, e.g., Chou et al., supra; Li et al., Proc. Natl. Acad. Sci. USA87:4580 (1990)). Accordingly, inhibition of DNA polymerase activity atambient temperature is beneficial in controlling the generation ofsecondary amplicons.

Several “hot start” techniques have been described which reportedlydecrease the formation of undesired secondary amplification products.According to certain “manual hot start” techniques, a component criticalto DNA polymerase activity (e.g., divalent ions and/or the DNApolymerase itself) is not added to the reaction mixture until thetemperature of the mixture is high enough to prevent non-specific primerannealing (see, e.g., Chou et al., supra; D'Aquila et al., Nucl. AcidsRes. 19:3749 (1991)). Less labor-intensive techniques employ thephysical separation or reversible inactivation of at least one componentof the amplification reaction. For example, the magnesium or the DNApolymerase can be sequestered in a wax bead, which melts as the reactiontemperature increases, releasing the sequestered component only at theelevated temperature. According to other techniques, the DNA polymeraseis reversibly inactivated or modified, for example by a reversiblechemical modification of the DNA polymerase, the binding of an antibodyto the DNA polymerase, or oligonucleotide molecules that bind to the DNApolymerase (see, e.g., U.S. Pat. Nos. 5,677,152 and 5,338,671; and Danget al., J. Mol. Biol. 264:268 (1996)). At an elevated reactiontemperature, the chemical modification is reversed, or the antibodymolecule or oligonucleotide molecule is denatured, releasing afunctional DNA polymerase. However, some of these techniques appear tobe less than optimal, in that some DNA polymerase activity is detectableat lower reaction temperatures despite the inactivation, or they requireextended exposure of the reaction mixture at high temperatures to fullyactivate the DNA polymerase, which may result in permanent inactivationof some components of the reaction mixture.

Certain currently used nucleic acid amplification techniques include astep for detecting and/or quantifying amplification products thatcomprise a nucleic acid dye, for example, but not limited to, SYBR®Green I (Life Technologies, Carlsbad, Calif.), including certainreal-time and/or end-point detection techniques (see, e.g., Ririe etal., Anal. Biochem. 245:154 (1997)). Typically the nucleic acid dyeassociates with double-stranded segments of the amplification productsand/or primer-template duplexes and emits a detectable fluorescentsignal at a wavelength that is characteristic of the particular nucleicacid dye. Certain amplification methods comprise a detection step forevaluating the purity of the amplification product(s) associated withthe nucleic acid dye, for example but not limited to, post-PCRdissociation curve analysis, also known as melting curve analysis. Sincethe melting curve of an amplicon is dependent on its length and sequence(among other things), amplicons can generally be distinguished by theirmelting curves (see, e.g., Zhang et al., Hepatology 36:723 (2002)). Adissociation or melting curve can be obtained during certainamplification reactions by monitoring the nucleic acid dye fluorescenceas the reaction temperatures pass through the melting temperature of theamplicon(s). The dissociation of a double-stranded amplicon is observedas a sudden decrease in fluorescence at the emission wavelengthcharacteristic of the nucleic acid dye. According to certaindissociation curve analysis techniques, an amplification product isclassified as “pure” when the melting curve shows a single, consistentmelting temperature, sometimes graphically displayed as a peak on a plotof the negative derivative of fluorescent intensity versus temperature(−dF/dt vs. T). In contrast, the appearance of multiple peaks in such adissociation curve from a single-plex amplification typically indicatesthe presence of undesired side reaction products. When such nucleic aciddye-based amplification product detection techniques are employed, it isoften desirable to: 1) at least decrease and preferably eliminate theformation of undesired side-reaction products, and 2) at least decreaseand preferably eliminate fluorescence peaks resulting from thedenaturing of double-stranded segments of other nucleic acids, i.e.,non-amplification products (e.g., primer dimers) and/or non-specificamplification products (e.g., due to mis-priming events).

Certain other amplification techniques may also yield undesiredamplification products due to, among other things, non-specificannealing of primers, ligation probes, cleavage probes,promoter-primers, and so forth, and subsequent enzyme activity atsub-optimal temperatures. For example, while reaction components arebeing combined, often at room temperature, or while the reactioncomposition is being heated to a desired reaction temperature. At leastsome of these techniques can benefit from a reduction in backgroundfluorescence. Thus, there is a need for compositions and methods thatdecrease and/or eliminate 1) the formation of undesired side-reactionproducts and 2) the background fluorescence resulting from theseundesired side-reaction products.

SUMMARY

The present teachings are directed to compositions, methods and kits foramplifying target nucleic acids while reducing non-specificamplification (e.g., fluorescence) and undesired amplification products,sometimes referred to in the art as secondary amplicons or spuriousside-products.

In certain embodiments, compositions are provided that comprise anucleic acid polymerase and a dual hot start reaction mixture thatinhibits or substantially inhibits the polymerase activity of thenucleic acid polymerase at a first temperature (e.g., <40° C.temperature). The dual hot start reaction mixture comprises at least twodifferent hot start mechanisms that are used to inhibit or substantiallyinhibit the polymerase activity of a nucleic acid polymerase at a firsttemperature. Such hot start mechanisms include, but are not limited to,antibodies or combinations of antibodies that block DNA polymeraseactivity at lower temperatures, oligonucleotides that block DNApolymerase activity at lower temperatures, reversible chemicalmodifications of the DNA polymerase that dissociate at elevatedtemperatures, amino acid modifications of the DNA polymerase thatprovide reduced activity at lower temperatures, fusion proteins thatinclude hyperstable DNA binding domains and topoisomerase, temperaturedependent ligands that inhibit the DNA polymerase, single strandedbinding proteins that sequester primers at lower temperatures, modifiedprimers or modified dNTPs.

In certain embodiments, the dual hot start reaction mixture inhibitsnon-specific nucleic acid amplification and/or non-specific productformation for extended periods of time compared to conventional hotstart mechanisms. For example, in some embodiments, the present dual hotstart reaction mixture inhibits non-specific nucleic acid amplificationand/or non-specific product formation for at least 24 hours at ambienttemperature. In certain embodiments, the dual hot start reaction mixturedecreases non-specific amplification and/or non-specific productformation by about 20-100% as compared to a hot start reaction mixturehaving only a single hot start mechanism. In certain embodiments, thedual hot start reaction mixture decreases non-specific amplificationand/or non-specific product formation by about 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments,the dual hot start reaction mixture reduces non-specific productformation by about 2- to 4-fold as compared to a reaction mixture havingonly a single hot start mechanism. In certain embodiments, the dual hotstart reaction mixture reduces non-specific product formation by about2-, 2.5-, 3-, 3.5-, or 4-fold.

In certain embodiments, compositions are provided that comprise athermostable nucleic acid polymerase and a dual hot start reactionmixture that inhibits or substantially inhibits the polymerase activityof the nucleic acid polymerase at a temperature less than about 40° C.and such that the dual hot start reaction mixture does not substantiallyinhibit the polymerase activity of the nucleic acid polymerase at atemperature greater than about 40° C. In certain embodiments, thenucleic acid polymerase may be a DNA-dependent DNA polymerase or anRNA-dependent DNA polymerase. In certain embodiments, the nucleic acidpolymerase may be thermostable.

In certain embodiments, methods for inhibiting the polymerase activityof a nucleic acid polymerase are provided. In certain embodiments, thesemethods involve contacting the polymerase with a dual hot start reactionmixture, where the dual hot start reaction mixture inhibits orsubstantially inhibits the polymerase activity of the nucleic acidpolymerase at a first temperature (e.g., <40° C. temperature).Polymerase inhibition may be reversible (e.g., by heating to atemperature greater than said first temperature, typically to atemperature of at least about 40° C.). The dual hot start reactionmixture comprises at least two different hot start mechanisms that areused to inhibit or substantially inhibit the polymerase activity of anucleic acid polymerase at a first temperature (e.g., ambienttemperature). Such hot start mechanisms include, but are not limited to,antibodies or combinations of antibodies that block DNA polymeraseactivity at lower temperatures, oligonucleotides that block DNApolymerase activity at lower temperatures, reversible chemicalmodifications of the DNA polymerase that dissociate at elevatedtemperatures, amino acid modifications of the DNA polymerase thatprovide reduced activity at lower temperatures, fusion proteins thatinclude hyperstable DNA binding domains and topoisomerase, temperaturedependent ligands that inhibit the DNA polymerase, single strandedbinding proteins that sequester primers at lower temperatures, modifiedprimers, or modified dNTPs. In certain embodiments, the nucleic acidpolymerase may be a DNA-dependent DNA polymerase or an RNA-dependent DNApolymerase. In certain embodiments, the nucleic acid polymerase may bethermostable.

In certain embodiments, methods for synthesizing a nucleic acid moleculeare provided. Such methods involve contacting a template nucleic acidwith a composition comprising a thermostable nucleic acid polymerase, adual hot start reaction mixture, one or more nucleoside and/ordeoxynucleoside triphosphates and at least one primer, wherein the dualhot start reaction mixture inhibits or substantially inhibits polymeraseactivity of the nucleic acid polymerase (compared polymerase activity inreaction mixtures without a dual hot start mechanism) at a lowertemperature (e.g., ambient temperature), bringing the resulting mixtureto a higher temperature sufficient to relieve polymerase inhibition, andpolymerizing the template nucleic acid.

In certain embodiments, methods for reducing non-specific fluorescenceusing the dual hot start reaction mixture are provided. According tosuch methods, a nucleic acid polymerase is contacted with the dual hotstart reaction mixture at a first temperature under conditions suitablefor the dual hot start reaction mixture to substantially inhibitpolymerase activity of the nucleic acid polymerase. When the resultingmixture is heated to a suitable second temperature, the dual hot startreaction mixture halts inhibition of the nucleic acid polymerase ornucleic acid polymerase activity.

In certain embodiments, the dual hot start reaction mixture inhibitsnon-specific nucleic acid amplification and/or non-specific productformation for extended periods of time compared to conventional hotstart mechanisms. For example, in some embodiments, the present dual hotstart reaction mixture inhibits non-specific nucleic acid amplificationand/or non-specific product formation for at least 24 hours at ambienttemperature. In certain embodiments, the dual hot start reaction mixturedecreases non-specific amplification and/or non-specific productformation by about 20-100% as compared to a hot start reaction mixturehaving only a single hot start mechanism. In certain embodiments, thedual hot start reaction mixture decreases non-specific amplificationand/or non-specific product formation by about 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments,the dual hot start reaction mixture reduces non-specific productformation by about 2- to 4-fold as compared to a reaction mixture havingonly a single hot start mechanism. In certain embodiments, the dual hotstart reaction mixture reduces non-specific product formation by about2-, 2.5-, 3-, 3.5-, or 4-fold.

In certain embodiments, methods for reducing non-specific fluorescenceusing a dual hot start reaction mixture are provided. According to suchmethods, a reaction composition is formed at a first temperaturecomprising a nucleic acid polymerase, a dual hot start reaction mixture,at least one NTP or dNTP, a target nucleic acid, at least one primer andat least one nucleic acid binding dye. In certain embodiments, the atleast one primer comprises a primer pair. At the first temperature(e.g., <40° C. temperature, such as ambient or room temperature), thedual hot start reaction mixture inhibits or substantially inhibitspolymerase activity of the nucleic acid polymerase. The reactioncomposition is subsequently heated to a second reaction temperature thatcauses the dual hot start reaction mixture to restore activity to thenucleic acid polymerase. The reaction composition is subjected to atleast one cycle of amplification and at least one amplicon is generated.The double-stranded amplicons may be detected, either in “real time” orafter the amplification reaction is completed due to the fluorescence ofthe nucleic acid binding dye associated with the amplicons.

In certain embodiments, the dual hot start reaction mixture inhibitsnon-specific nucleic acid amplification and/or non-specific productformation for extended periods of time compared to conventional hotstart mechanisms. For example, in some embodiments, the present dual hotstart reaction mixture inhibits non-specific nucleic acid amplificationand/or non-specific product formation for at least 24 hours at ambienttemperature. In certain embodiments, the dual hot start reaction mixturedecreases non-specific amplification and/or non-specific productformation by about 20-100% as compared to a hot start reaction mixturehaving only a single hot start mechanism. In certain embodiments, thedual hot start reaction mixture decreases non-specific amplificationand/or non-specific product formation by about 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments,the dual hot start reaction mixture reduces non-specific productformation by about 2- to 4-fold as compared to a reaction mixture havingonly a single hot start mechanism. In certain embodiments, the dual hotstart reaction mixture reduces non-specific product formation by about2-, 2.5-, 3-, 3.5-, or 4-fold.

In certain embodiments, methods for amplifying a target nucleic acidusing the dual hot start reaction mixture are provided. According tocertain such methods, a reaction composition is formed at a firsttemperature comprising a nucleic acid polymerase, a dual host startreaction mixture, at least one NTP or dNTP, a target nucleic acid, atleast one primer and at least one nucleic acid binding dye. In certainembodiments, the at least one primer comprises a primer pair. At thefirst temperature, the dual hot start reaction mixture inhibits orsubstantially inhibits polymerase activity of the nucleic acidpolymerase. The reaction composition is subsequently heated to a secondreaction temperature that causes the dual hot start reaction mixture tohalt inhibition of the nucleic acid polymerase or nucleic acidpolymerase activity. The reaction composition is subjected to at leastone cycle of amplification and a multiplicity of amplicons is generated.The double-stranded amplicons may be detected, either in “real time” orafter the amplification reaction is completed due to the fluorescence ofthe nucleic acid binding dye associated with the amplicons.

In certain embodiments, kits for performing certain of the instantmethods are also provided. In certain embodiments, the kits comprise adual hot start reaction mixture. In certain embodiments, the kitsfurther comprise at least one nucleic acid polymerase. In certainembodiments, the kits further comprise one or more of: at least oneprimer or a primer pair, a nucleic acid binding dye, a reporter probe,and a reverse transcriptase.

These and other features of the present teachings are provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1D: Comparison of Fast SYBR® Green Master Mix alone(“square” line), Platinum® SYBR® Green qPCR Master Mix alone (“cross”line) and Fast SYBR® Green Master Mix in combination with Platinum®SYBR® Green qPCR Master Mix at a 1:1 ratio (“triangle” line). FIG. 1A:Amplification of cDNA at 0 h room temperature pre-incubation (“T0”);FIG. 1B: Amplification of NTC (non-specific products) at T0; C:Amplification of cDNA at 24 h room temperature pre-incubation (“T24”);and D: Amplification of NTC at T24.

FIGS. 2A-1 through 2N: Nucleic acid amplification using Fast SYBR® GreenMaster Mix and Platinum® SYBR® Green qPCR Master Mix, alone or incombination. FIG. 2A-1 through 2C-2: Melt curve analysis of A2M cDNA andNTC at T0; FIG. 2D-1 through 2F-2: Melt curve analysis of A2M cDNA andNTC at T24; FIG. 2G-1 through 2I-2: Melt curve analysis of COL1A1 cDNAand NTC at T0; FIG. 2J-1 through 2L-2: Melt curve analysis of COL1A1cDNA and NTC at T24; FIG. 2M: Comparison of mean Ct and ΔCt for FastSYBR® Green Master Mix and Platinum® SYBR® Green qPCR Master Mix, aloneor in combination; FIG. 2N: Melt curve analysis of Fast SYBR® GreenMaster Mix and Platinum® SYBR® Green qPCR Master Mix, alone or incombination.

FIGS. 3A through 3D: Comparison of Fast SYBR® Green Master Mix alone(“diamond” line) and combination of Fast SYBR® Green Master Mix andPlatinum® Taq Antibody (“cross” line). FIG. 3A: Amplification of cDNA atT0; FIG. 3B: Amplification of NTC at T0; FIG. 3C: Amplification of cDNAat T24; and FIG. 3D: Amplification of NTC at T24.

FIGS. 4A-1 through 4F: Nucleic acid amplification using Fast SYBR® GreenMaster Mix and Platinum® Taq Antibody, alone or in combination. FIG.4A-1 through 4B-2: Melt curve analysis of A2M using Fast SYBR® GreenMaster Mix alone at T0 and T24; FIG. 4C-1 through 4E: Melt curveanalysis of A2M using a combination of Fast SYBR® Green Master Mix andPlatinum® Taq Antibody at T0 and T24; FIG. 4F: Melt curve analysis ofFast SYBR® Green Master Mix and Platinum® Taq Antibody, alone or incombination.

FIGS. 5A through 5D: Comparison of Power SYBR® Green PCR Master Mixalone (“diamond” line) and combination of Power SYBR® Green PCR Mix andPlatinum® Taq Antibody (“cross” line). FIG. 5A: Amplification of cDNA atT0; FIG. 5B: Amplification of NTC at T0; FIG. 5C: Amplification of cDNAat T24; and FIG. 5D: Amplification of NTC at T24.

FIGS. 6A-1 through 6E: Nucleic acid amplification using Power SYBR®Green PCR Master Mix and Platinum® Taq Antibody, alone or incombination. FIG. 6A-1 through 6B-2: Melt curve analysis of A2M usingPower SYBR® Green PCR Master Mix alone at T0 and T24; FIG. 6C-1 through6D-2: Melt curve analysis of A2M using a combination of Power SYBR®Green PCR Master Mix and Platinum® Taq Antibody at T0 and T24; FIG. 6E:Melt curve analysis of Power SYBR® Green PCR Master Mix and Platinum®Taq Antibody, alone or in combination.

DETAILED DESCRIPTION

In certain embodiments, compositions are provided that comprise anucleic acid polymerase and a dual hot start reaction mixture thatinhibits or substantially inhibits the polymerase activity of thenucleic acid polymerase at a first temperature (e.g., <40° C.temperature). In some embodiments, the dual hot start reaction mixturecomprises at least two different hot start mechanisms that are used toinhibit or substantially inhibit the polymerase activity of a nucleicacid polymerase at a first (e.g., lower) temperature. Such hot startmechanisms can include, for example, but are not limited to, antibodiesor combinations of antibodies that block DNA polymerase activity atlower temperatures, oligonucleotides that block DNA polymerase activityat lower temperatures, reversible chemical modifications of the DNApolymerase that dissociate at elevated temperatures, amino acidmodifications of the DNA polymerase that provide reduced activity atlower temperatures, fusion proteins that include hyperstable DNA bindingdomains and topoisomerase, temperature dependent ligands that inhibitthe DNA polymerase, single stranded binding proteins that sequesterprimers at lower temperatures, modified primers, or modified dNTPs.

In certain embodiments, the dual hot start reaction mixture inhibitsnon-specific nucleic acid amplification and/or non-specific productformation for extended periods of time compared to conventional hotstart mechanisms. For example, in some embodiments, the present dual hotstart reaction mixture inhibits non-specific nucleic acid amplificationand/or non-specific product formation for at least 24 hours at ambienttemperature. In certain embodiments, the dual hot start reaction mixturedecreases non-specific amplification and/or non-specific productformation by about 20-100% as compared to a hot start reaction mixturehaving only a single hot start mechanism. In certain embodiments, thedual hot start reaction mixture decreases non-specific amplificationand/or non-specific product formation by about 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments,the dual hot start reaction mixture reduces non-specific productformation by about 2- to 4-fold as compared to a reaction mixture havingonly a single hot start mechanism. In certain embodiments, the dual hotstart reaction mixture reduces non-specific product formation by about2-, 2.5-, 3-, 3.5-, or 4-fold.

In certain embodiments, compositions are provided that comprise athermostable nucleic acid polymerase and a dual hot start reactionmixture that inhibits or substantially inhibits the polymerase activityof the nucleic acid polymerase at a temperature less than about 40° C.and such that the dual hot start reaction mixture does not substantiallyinhibit the polymerase activity of the nucleic acid polymerase at atemperature greater than about 40° C. In certain embodiments, thenucleic acid polymerase may be a DNA-dependent DNA polymerase or anRNA-dependent DNA polymerase. In certain embodiments, the nucleic acidpolymerase may be thermostable.

In certain embodiments, methods for inhibiting the polymerase activityof a nucleic acid polymerase are provided. These methods involvecontacting the polymerase with a dual hot start reaction mixture, wherethe dual hot start reaction mixture inhibits or substantially inhibitsthe polymerase activity of the nucleic acid polymerase at a firsttemperature (e.g., <40° C. temperature). Polymerase inhibition may bereversible (e.g., by heating to a second temperature of at least about40° C.). The dual hot start reaction mixture comprises at least twodifferent hot start mechanisms that are used to inhibit or substantiallyinhibit the polymerase activity of a nucleic acid polymerase at a firsttemperature. Such hot start mechanisms include, but are not limited to,antibodies or combinations of antibodies that block DNA polymeraseactivity at lower temperatures, oligonucleotides that block DNApolymerase activity at lower temperatures, reversible chemicalmodifications of the DNA polymerase that dissociate at elevatedtemperatures, amino acid modifications of the DNA polymerase thatprovide reduced activity at lower temperatures, fusion proteins thatinclude hyperstable DNA binding domains and topoisomerase, temperaturedependent ligands that inhibit the DNA polymerase, single strandedbinding proteins that sequester primers at lower temperatures, modifiedprimers, or modified dNTPs. In certain embodiments, the nucleic acidpolymerase may be a DNA-dependent DNA polymerase or an RNA-dependent DNApolymerase. In certain embodiments, the nucleic acid polymerase may bethermostable.

In certain embodiments, methods for synthesizing a nucleic acid moleculeare provided. Such methods involve contacting a template nucleic acidwith a composition comprising a thermostable nucleic acid polymerase, adual hot start reaction mixture, one or more nucleoside and/ordeoxynucleoside triphosphates and at least one primer, wherein the dualhot start reaction mixture inhibits or substantially inhibits polymeraseactivity of the nucleic acid polymerase at first temperature, bringingthe resulting mixture to a second temperature sufficient to relievepolymerase inhibition, and polymerizing the template nucleic acid. Insome embodiments, the first temperature is an ambient temperature (e.g.,room temperature). In certain embodiments, the nucleic acid polymerasemay be a DNA-dependent DNA polymerase or an RNA-dependent DNApolymerase. In certain embodiments, the nucleic acid polymerase may bethermostable.

In certain embodiments, methods for preventing or reducing mis-primingevents comprising a dual hot start reaction mixture are provided.According to such methods, a nucleic acid polymerase is contacted withthe dual hot start reaction mixture at a first temperature underconditions suitable for the dual hot start reaction mixture tosubstantially inhibit polymerase activity of the nucleic acidpolymerase. When the resulting mixture is heated to a suitable secondtemperature, the dual hot start mechanism is halted from inhibiting thepolymerase or polymerase activity and primer extension is allowed tooccur.

In certain embodiments, methods for reducing non-specific amplificationproduct formation comprising a dual hot start reaction mixture areprovided. According to such methods, a target nucleic acid is contactedwith a nucleic acid polymerase, a dual hot start reaction mixture, atleast one primer and at least one dNTP. In certain embodiments, the atleast one primer comprises a primer pair. At the first temperature, thedual hot start reaction mixture inhibits or substantially inhibitspolymerase activity of the nucleic acid polymerase. The reactioncomposition is subsequently heated to a second temperature that preventsthe dual hot start reaction from inhibiting the nucleic acid polymerase.The target nucleic acid in the reaction composition can then besubjected to at least one cycle of amplification. In certain embodimentsthe nucleic acid polymerase is thermostable. In certain otherembodiments the nucleic acid polymerase is a DNA-dependent DNApolymerase or an RNA-dependent DNA polymerase. For example, the nucleicacid polymerase may be selected from the group consisting of, but notlimited to, Taq DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase,Pfu DNA polymerase, and Vent™ DNA polymerase. In some other embodiments,the dual hot start reaction mixture comprises at least two different hotstart mechanisms. In some embodiments, the at least two different hotstart mechanisms can be selected from the group consisting of antibodiesor combinations of antibodies that block DNA polymerase activity atlower temperatures, oligonucleotides that block DNA polymerase activityat lower temperatures, reversible chemical modifications of the DNApolymerase that dissociate at elevated temperatures, amino acidmodifications of the DNA polymerase that provide reduced activity atlower temperatures, fusion proteins that include hyperstable DNA bindingdomains and topoisomerase, temperature dependent ligands that inhibitthe DNA polymerase, single stranded binding proteins that sequesterprimers at lower temperatures, modified primers, or modified dNTPs.

In certain embodiments, the dual hot start reaction mixture providesincreased inhibition of non-specific nucleic acid amplification and/ornon-specific product formation compared to conventional hot startmechanisms. In other embodiments, the dual hot start reaction mixtureinhibits non-specific nucleic acid amplification and/or non-specificproduct formation for extended periods of time compared to conventionalhot start mechanisms. For example, in some embodiments, the dual hotstart reaction mixture inhibits non-specific nucleic acid amplificationand/or non-specific product formation for at least 24 hours at ambienttemperature. In certain embodiments, the dual hot start reaction mixturedecreases non-specific amplification and/or non-specific productformation by about 20-100% as compared to a hot start reaction mixturehaving only a single hot start mechanism. In certain embodiments, thedual hot start reaction mixture decreases non-specific amplificationand/or non-specific product formation by about 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments,the dual hot start reaction mixture reduces non-specific productformation by about 2- to 4-fold as compared to a reaction mixture havingonly a single hot start mechanism. In certain embodiments, the dual hotstart reaction mixture reduces non-specific product formation by about2-, 2.5-, 3-, 3.5-, or 4-fold.

In certain embodiments, methods for reducing non-specific fluorescencecomprise a dual hot start reaction mixture are provided. According tosuch methods, a reaction composition is formed at a first temperaturecomprising a nucleic acid polymerase, a dual host start reactionmixture, at least one NTP or dNTP, a target nucleic acid, at least oneprimer and at least one nucleic acid binding dye. In certainembodiments, the at least one primer comprises a primer pair. At thefirst temperature, the dual hot start reaction mixture inhibits orsubstantially inhibits polymerase activity of the nucleic acidpolymerase. The reaction composition is subsequently heated to a secondreaction temperature that causes the dual hot start reaction mixture toallow nucleic acid polymerase activity to occur. The reactioncomposition is subjected to at least one cycle of amplification and amultiplicity of amplicons is generated. The double-stranded ampliconsmay be detected, either in “real time” or after the amplificationreaction is completed due to the fluorescence of the nucleic acidbinding dye associated with the amplicons.

In certain embodiments, the dual hot start reaction mixture inhibitsnon-specific nucleic acid amplification and/or non-specific productformation for extended periods of time compared to conventional hotstart mechanisms. For example, in some embodiments, the present dual hotstart reaction mixture inhibits non-specific nucleic acid amplificationand/or non-specific product formation for at least 24 hours at ambienttemperature. In certain embodiments, the dual hot start reaction mixturedecreases non-specific amplification and/or non-specific productformation by about 20-100% as compared to a hot start reaction mixturehaving only a single hot start mechanism. In certain embodiments, thedual hot start reaction mixture decreases non-specific amplificationand/or non-specific product formation by about 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments,the dual hot start reaction mixture reduces non-specific productformation by about 2- to 4-fold as compared to a reaction mixture havingonly a single hot start mechanism. In certain embodiments, the dual hotstart reaction mixture reduces non-specific product formation by about2-, 2.5-, 3-, 3.5-, or 4-fold.

In certain embodiments, methods for amplifying a target nucleic acidusing the dual hot start reaction mixture are provided. According tocertain such methods, a reaction composition is formed at a firsttemperature comprising a nucleic acid polymerase, a dual host startreaction mixture, at least one NTP or dNTP, a target nucleic acid, atleast one primer and at least one nucleic acid binding dye. In certainembodiments, the at least one primer comprises a primer pair. At thefirst temperature, the dual hot start reaction mixture inhibits orsubstantially inhibits polymerase activity of the nucleic acidpolymerase. The reaction composition is subsequently heated to a secondreaction temperature that causes the dual hot start reaction mixture tohalt inhibition of the nucleic acid polymerase or nucleic acidpolymerase activity. The reaction composition is subjected to at leastone cycle of amplification and a multiplicity of amplicons is generated.The double-stranded amplicons may be detected, either in “real time” orafter the amplification reaction is completed due to the fluorescence ofthe nucleic acid binding dye associated with the amplicons.

In certain embodiments, kits for performing certain of the instantmethods are also provided. In certain embodiments, the kits comprise adual hot start reaction mixture. In certain embodiments, the kitsfurther comprise at least one nucleic acid polymerase. In certainembodiments, the kits further comprise one or more of: at least oneprimer or a primer pair, a nucleic acid binding dye, a reporter probe,and a reverse transcriptase.

To more clearly and concisely describe and point out the subject matterof the present disclosure, the following definitions are provided forspecific terms, which are used in the following description and theappended claims. Throughout the specification, exemplification ofspecific terms should be considered as non-limiting examples.

As used in this specification, the words “a” or “an” means at least one,unless specifically stated otherwise. In this specification, the use ofthe singular includes the plural unless specifically stated otherwise.For example, but not as a limitation, “a target nucleic acid” means thatmore than one target nucleic acid can be present; for example, one ormore copies of a particular target nucleic acid species, as well as twoor more different species of target nucleic acid. The term “and/or”means that the terms before and after the slash can be taken together orseparately. For illustration purposes, but not as a limitation, “Xand/or Y” can mean “X” or “Y” or “X” and “Y”.

It will be appreciated that there is an implied “about” prior to thetemperatures, concentrations, times, etc. discussed in the presentdisclosure, such that slight and insubstantial deviations are within thescope of the present teachings herein. Also, the use of “comprise”,“comprises”, “comprising”, “contain”, “contains”, “containing”,“include”, “includes”, and “including” are not intended to be limiting.It is to be understood that both the foregoing general description anddetailed description are exemplary and explanatory only and are notrestrictive of the teachings.

Unless specifically noted in the above specification, embodiments in theabove specification that recite “comprising” various components are alsocontemplated as “consisting of” or “consisting essentially of” therecited components; embodiments in the specification that recite“consisting of” various components are also contemplated as “comprising”or “consisting essentially of” the recited components; and embodimentsin the specification that recite “consisting essentially of” variouscomponents are also contemplated as “consisting of” or “comprising” therecited components (this interchangeability does not apply to the use ofthese terms in the claims).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the desired subject matter inany way. All literature cited in the specification, including but notlimited to, patent, patent applications, articles, books and treatisesare expressly incorporated by reference in their entirety for anypurpose. In the event that any of the incorporated literaturecontradicts any term defined in this specification, this specificationcontrols. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

The terms “amplicon” and “amplification product” as used hereingenerally refer to the product of an amplification reaction. An ampliconmay be double-stranded or single-stranded, and may include the separatedcomponent strands obtained by denaturing a double-stranded amplificationproduct. In certain embodiments, the amplicon of one amplification cyclecan serve as a template in a subsequent amplification cycle.

The terms “annealing” and “hybridizing”, including, without limitation,variations of the root words “hybridize” and “anneal”, are usedinterchangeably and mean the nucleotide base-pairing interaction of onenucleic acid with another nucleic acid that results in the formation ofa duplex, triplex, or other higher-ordered structure. The primaryinteraction is typically nucleotide base specific, e.g., A:T, A:U, andG:C, by Watson-Crick and Hoogsteen-type hydrogen bonding. In certainembodiments, base-stacking and hydrophobic interactions may alsocontribute to duplex stability. Conditions under which primers andprobes anneal to complementary sequences are well known in the art,e.g., as described in Nucleic Acid Hybridization, A Practical Approach,Hames and Higgins, eds., IRL Press, Washington, D.C. (1985) and Wetmurand Davidson, Mol. Biol. 31:349 (1968).

In general, whether such annealing takes place is influenced by, amongother things, the length of the complementary portions of thecomplementary portions of the primers and their corresponding bindingsites in the target flanking sequences and/or amplicons, or thecorresponding complementary portions of a reporter probe and its bindingsite; the pH; the temperature; the presence of mono- and divalentcations; the proportion of G and C nucleotides in the hybridizingregion; the viscosity of the medium; and the presence of denaturants.Such variables influence the time required for hybridization. Thus, thepreferred annealing conditions will depend upon the particularapplication. Such conditions, however, can be routinely determined bypersons of ordinary skill in the art, without undue experimentation.Preferably, annealing conditions are selected to allow the primersand/or probes to selectively hybridize with a complementary sequence inthe corresponding target flanking sequence or amplicon, but nothybridize to any significant degree to different target nucleic acids ornon-target sequences in the reaction composition at the second reactiontemperature.

The term “selectively hybridize” and variations thereof, means that,under appropriate stringency conditions, a given sequence (for example,but not limited to a primer) anneals with a second sequence comprising acomplementary string of nucleotides (for example, but not limited to atarget flanking sequence or primer binding site of an amplicon), butdoes not anneal to undesired sequences, such as non-target nucleicacids, probes, or other primers. Typically, as the reaction temperatureincreases toward the melting temperature of a particular double-strandedsequence, the relative amount of selective hybridization generallyincreases and mis-priming generally decreases. In this specification, astatement that one sequence hybridizes or selectively hybridizes withanother sequence encompasses situations where the entirety of both ofthe sequences hybridize or selectively hybridize to one another, andsituations where only a portion of one or both of the sequenceshybridizes or selectively hybridizes to the entire other sequence or toa portion of the other sequence.

As used herein, the term “stringency” is used to define the temperatureand solvent composition existing during hybridization and the subsequentprocessing steps at which a hybrid comprised of two complementarynucleotide sequences will form. Stringency also defines the amount ofhomology, the conditions necessary, and the stability of hybrids formedbetween two nucleotide sequences. As the stringency conditions increase,selective hybridization is favored and non-specific cross-hybridizationis disfavored. Increased stringency conditions typically correspond tohigher incubation temperature, lower salt concentrations, and/or higherpH, relative to lower stringency conditions at which mis-priming is morelikely to occur. Those in the art understand that appropriate stringencyconditions to enable the selective hybridization of a primer or primerpair to a corresponding target flanking sequence and/or amplicon can beroutinely determined using well known techniques and without undueexperimentation (see, e.g., PCR: The Basics from Background to Bench,McPherson and Moller, Bios Scientific Publishers, 2000).

As used herein, dual hot start reaction mixtures or mechanisms that“substantially inhibit” polymerase activity refers to reaction mixturesthat provide less than about 30%, less than about 25%, less than about20%, more preferably less than about 15%, less than about 10%, less thanabout 7.5%, or less than about 5%, and most preferably less than about5%, less than about 2%, less than about 1%, less than about 0.5%, lessthan about 0.25% polymerase activity, or which lack polymerase activityaltogether. Polymerase activity that is “substantially inhibited” asused herein refers to polymerase activity that is at least about 70%,75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.75%, 100% or >100% inhibited inthe presence of said hot start mechanisms or hot start reactionmixtures.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed terms preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

The terms “denaturing” and “denaturation” as used herein refer to anyprocess in which a double-stranded polynucleotide, including withoutlimitation, a genomic DNA (gDNA) fragment comprising at least one targetnucleic acid, a double-stranded amplicon, or a polynucleotide comprisingat least one double-stranded segment is converted to two single-strandedpolynucleotides or to a single-stranded or substantially single-strandedpolynucleotide, as appropriate. Denaturing a double-strandedpolynucleotide includes, without limitation, a variety of thermal andchemical techniques which render a double-stranded nucleic acidsingle-stranded or substantially single-stranded, for example but notlimited to, releasing the two individual single-stranded components of adouble-stranded polynucleotide or a duplex comprising twooligonucleotides. Those in the art will appreciate that the denaturingtechnique employed is generally not limiting unless it substantiallyinterferes with a subsequent annealing or enzymatic step of anamplification reaction, or in certain methods, the detection of afluorescent signal.

As used herein, the term “Tm” is used in reference to meltingtemperature. The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands.

The term “minor groove binder” as used herein refers to a small moleculethat fits into the minor groove of double-stranded DNA, sometimes in asequence specific manner. Generally, minor groove binders are long, flatmolecules that can adopt a crescent-like shape and thus, fit snugly intothe minor groove of a double helix, often displacing water. Minor groovebinding molecules typically comprise several aromatic rings connected bybonds with torsional freedom, for example, but not limited to, furan,benzene, or pyrrole rings.

“Mis-priming” or “mis-primed” as used herein, refer to the hybridizationof a primer or a probe to a non-target nucleic acid. As is known in theart, primers (excluding random primers) are generally designed tohybridize to a selected sequence that flanks a target nucleic acid or toa primer binding site of an amplicon and to direct DNA synthesis orprimer extension starting at that site. Mis-priming may occur when aprimer or a probe hybridizes to a non-target nucleic acid, oftentimes atlow or decreased stringency conditions, and then serves as theinitiation point for primer extension from that non-target site, givingrise to synthesis of certain undesired secondary amplification products.

The terms “non-specific” or “background” when used in reference tofluorescence refers to the detectable signal emitted from nucleic acidbinding dye molecules associated with double-stranded nucleic acidsother than desired amplicons. Desired amplicons comprise theamplification products of target nucleic acids, including in someembodiments, internal standard or control sequences that may be includedin certain reaction compositions of the current teachings for, amongother things, normalization and/or quantitation purposes. Thus, thefluorescent signal resulting from the association of nucleic acid dyemolecules with spurious, secondary amplicons, often the result ofmis-priming, mis-ligation, and/or primer-dimer formation, is one sourceof non-specific fluorescence.

The term “nucleic acid binding dye” as used herein refers to afluorescent molecule that is specific for a double-strandedpolynucleotide or that at least shows a substantially greaterfluorescent enhancement when associated with double-strandedpolynucleotides than with a single stranded polynucleotide. Typically,nucleic acid binding dye molecules associate with double-strandedsegments of polynucleotides by intercalating between the base pairs ofthe double-stranded segment, but binding in the major or minor groovesof the double-stranded segment, or both. Non-limiting examples ofnucleic acid binding dyes include ethidium bromide, DAPI, Hoechstderivatives including without limitation Hoechst 33258 and Hoechst33342, intercalators comprising a lanthanide chelate (for example, butnot limited to, a naphthalene diimide derivative carrying twofluorescent tetradentate β-diketone-Eu³⁺chelates (NDI-(BHHCT-Eu³⁺)₂),see e.g., Nojima et al., Nucl. Acids Res. Suppl. No. 1 105 (2001), anddertain unsymmetrical cyanine dyes such as SYBR® Green and PicoGreen®.

As used herein, the terms “polynucleotide”, “oligonucleotide,” and“nucleic acid” are used interchangeably and refer to single-stranded anddouble-stranded polymers of nucleotide monomers, including withoutlimitation, 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA)linked by internucleotide phosphodiester bond linkages, orinternucleotide analogs, and associated counter ions, e.g., H⁺, NH₄ ⁺,trialkylammonium, Mg²⁺, Na⁺, and the like. A polynucleotide may becomposed entirely of deoxyribonucleotides, entirely of ribonucleotides,or chimeric mixtures thereof and may include nucleotide analogs. Thenucleotide monomer units may comprise any of the nucleotides describedherein, including, but not limited to, nucleotides and/or nucleotideanalogs. Polynucleotides typically range in size from a few monomericunits, e.g., 5-40 when they are sometimes referred to in the art asoligonucleotides, to several thousands of monomeric nucleotide units.Unless denoted otherwise, whenever a polynucleotide sequence isrepresented, it will be understood that the nucleotides are in the5′-to-3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytosine, “G” denotes deoxyguanosine, “T” denotesdeoxythymidine, and “U” denotes deoxyuridine, unless otherwise noted.

The term “nucleotide” refers to a phosphate ester of a nucleoside, e.g.,triphosphate esters, wherein the most common site of esterification isthe hydroxyl group attached at the C-5 position of the pentose.

The term “nucleoside” refers to a compound consisting of a purine,deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine,cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like,linked to a pentose at the 1′ position, including 2′-deoxy and2′-hydroxyl forms. When the nucleoside base is purine or 7-deazapurine,the pentose is attached to the nucleobase at the 9-position of thepurine or deazapurine, and when the nucleobase is purimidine, thepentose is attached to the nucleobase at the 1-position of thepyrimidine.

The term “analog” includes synthetic analogs having modified basemoieties, modified sugar moieties, and/or modified phosphate estermoieties. Phosphate analogs generally comprise analogs of phosphatewherein the phosphorous atom is in the +5 oxidation state and one ormore of the oxygen atoms is replaced with a non-oxygen moiety, e.g.sulfur. Exemplary phosphate analogs include: phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate,boronophosphates, including associated counterions, e.g., H³⁰, NH₄ ⁺,Na⁺. Exemplary base analogs include: 2,6-diaminopurine, hypoxanthine,pseudouridine, C-5-propyne, isocytosine, isoguanine, 2-thiopyrimidine.Exemplary sugar analogs include: 2′- or 3′-modifications where the 2′-or 3′-position is hydrogen, hydroxy, alkoxy, e.g., methoxy, ethoxy,allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy, azido, amino oralkylamino, fluoro, chloro, and bromo.

As used herein, the term “reaction vessel” generally refers to anycontainer, chamber, device, or assembly, in which a reaction can occurin accordance with the present teachings. In some embodiments, areaction vessel may be a microtube, for example, but not limited to, a0.2 mL or a 0.5 mL reaction tube such as a MicroAmp® Optical tube (LifeTechnologies Corp., Carslbad, Calif.) or a micro-centrifuge tube, orother containers of the sort in common practice in molecular biologylaboratories. In some embodiments, a reaction vessel comprises a well ofa multi-well plate, a spot on a glass slide, or a channel or chamber ofa microfluidics device, including without limitation a TaqMan® LowDensity Array or a TaqMan® Open Array Real-Time PCR plate (both fromLife Technologies Corp.). For example, but not as a limitation, aplurality of reaction vessels can reside on the same support. In someembodiments, lab-on-a-chip-like devices available, for example, fromCaliper, Fluidigm and Life Technologies Corp., including the Ion 316™and Ion 318™ Chip, may serve as reaction vessels in the disclosedmethods. It will be recognized that a variety of reaction vessels arecommercially available or can be designed for use in the context of thepresent teachings.

As used herein, the term “hot start” generally refers to a means oflimiting the availability of an essential reaction component (e.g., apolymerase) when the reaction mixture is maintained at a firsttemperature (typically a lower temperature) until a second temperature(typically a higher temperature) is reached which allows the essentialcomponent to participate in the reaction. Hot start reactions typicallyinvolve incubation at a first (e.g., lower) temperature and subsequentelevation to a second (e.g., higher) temperature which allows thedesired reaction to take place. Activation of the hot start reaction ispreferably achieved by an incubation at a temperature which is equal toor higher than the primer hybridization (annealing) temperature used inthe amplification reaction to ensure primer binding specificity. Thelength of incubation required to recover enzyme activity depends on thetemperature and pH of the reaction mixture and on the stability of theenzyme. A wide range of incubation conditions are usable; optimalconditions may be determined empirically for each reaction. In general,a dual hot start reaction mixture is incubated at a first temperature toinhibit nucleic acid synthesis (e.g., polymerase activity) and thenelevated to a second temperature for inhibition to be relieved orhalted. Optimization of incubation conditions for the reactivation ofnucleic acid polymerases not exemplified, or for reaction mixtures notexemplified, can be determined by routine experimentation following theguidance provided herein.

In certain embodiments the first temperature is lower than said secondtemperature. In some embodiments the first temperature is <40° C. Incertain embodiments the first temperature is about equal to or less thanabout 40° C., 35° C., 30° C., 25° C., 20° C., 15° C., 10° C., 5° C., orless. In some embodiments, the first temperature is ambient temperature.In some other embodiments, the first temperature is room temperature. Insome embodiments the second temperature is >40° C. In certainembodiments the first temperature is about equal to or greater thanabout 40° C., 45° C., 50° C., 55° C., 60, 65° C., 70° C., 75° C., 80°C., 85° C., 90° C., 95° C., 100° C., or more. Several methods exist forperforming hot start reactions including, but not limited to, the use ofmanual techniques, barriers, chemical modifications and/or structuralmodifications of one or more of the essential reaction components.Suitable reaction conditions for such hot start methods are know in theart and further described herein and in the Examples.

As used herein, the term “dual hot start reaction mixture” refers to thecombination of reagents or reagent solutions which are used to blocknucleic acid polymerase extension at low temperatures (e.g., ambienttemperature) until the hot start conditions of the initial denaturationtemperature in an amplification reaction (e.g., PCR) are reached. At theelevated amplification temperature, the nucleic acid polymerase is nolonger inhibited and allows for primer extension. As used herein, thedual hot start reaction mixture is meant to include an reaction mixturethat comprises at least two different mechanisms for hot start.Accordingly, “dual hot start reaction mixtures” may include more thantwo hot start mechanisms (e.g., “triple hot start reaction mixture”,“quadruple hot start reaction mixture”, “quintuple hot start reactionmixture”, and so on). Possible hot start mechanisms may include, but arenot limited to, antibodies or combinations of antibodies that blocknucleic acid polymerase activity at lower temperatures and whichdissociate from the polymerase at elevated temperatures (see, e.g.,Eastlund et al., LifeSci. Quarterly 2:2 (2001), Mizuguchi et al., J.Biochem. (Tokyo) 126:762 (1999)); oligonucleotides that block nucleicacid polymerase activity at lower temperatures and which dissociate fromthe polymerase at elevated temperatures (see, e.g., Dang et al., J. Mol.Biol. 264:268 (1996)); reversibly chemical modification of the nucleicacid polymerase such that the nucleic acid polymerase activity isblocked at lower temperatures and the modifications reverse ordissociate at elevated temperatures (see, e.g., U.S. Pat. No. 5,773,258and Moretti et al., Biotechniques 25:716 (1998)); amino acid mutationsof the nucleic acid polymerase that provide reduced activity at lowertemperatures (see, e.g., Kermekchiev et al., Nucl. Acids Res. 31:6139(2003)); nucleic acid polymerase fusion proteins including hyperstableDNA binding domains and topoisomerases (see, e.g., Pavlov et al., Proc.Natl. Acad. Sci. USA 99:13510 (2002)); ligands that inhibit the nucleicacid polymerase in a temperature-dependent manner (for example,HotMaster™ Taq DNA polymerase from Eppendorf (Hauppauge, N.Y.) and 5PRIME (Gaithersburg, Md.)); single-stranded binding proteins thatsequester primers at low temperatures (see, e.g., U.S. PatentApplication Publication No. 2008/0138878); thermostable pyrophosphatasewhich hydrolyzes inorganic pyrophosphate at elevated temperatures (see,e.g., U.S. Patent Application Publication No. 2006/0057617);thermolabile blockers, such as a polymerase blocking protein (see, e.g.,U.S. Patent Application Publication No. 2007/0009922); primer competitorsequences (see, e.g., Puskas et al., Genome Res. 5:309 (1995) andVestheim et al., Front. Zool. 5:12 (2008)); modified primer constructs(see, e.g., Ailenberg et al., Biotechniques 29:22 (2000) and Kaboev etal., Nucl. Acids Res. 28:E94 (2000)); modified primers that improvehybridization selectivity (see, e.g., U.S. Pat. Nos. 6,794,142 and6,001,611); primers with 3′ modifications that are removable by 3′-5′exonuclease activity (see, e.g., U.S. Patent Application Publication No.2003/0119150 and U.S. Pat. No. 6,482,590); primers with modifiednucleobases that are removable by UV irradiation (see, e.g., Young etal., Chem. Commun. (Camb) 28:462 (2008)); primer modifications that areremovable by thermal deprotection (see, e.g., U.S. Patent ApplicationPublication No. 2003/0162199 and Lebedev et al., Nucl. Acids Res.36:e131 (2008)); or modification of the dNTPs with thermolabilemodification groups (see, e.g., U.S. Patent Application Publication No.2003/0162199 and Koukhareva et al., Nucl. Acids Symp. Ser. (Oxford), 259(2008)). All references cited herein are incorporated by reference intheir entirety for all purposes.

As used herein, dual hot start reaction mixtures comprising “at leasttwo different mechanisms” encompass those reaction mixtures that maycomprise at least two different hot start mechanisms that functionsimilarly or use similar components. For example, dual hot startreaction mixtures can comprise reagents or reagent solutions designedfor two different antibody-based hot start mechanisms, or two differentoligonucleotide-based hot start mechanisms, or one antibody-based andone oligonucleotide-based hot start mechanism, or one antibody-based andone chemical modification-based hot start mechanism, or any suchcombination available.

The term “reporter group” is used in a broad sense herein and refers toany identifiable tag, label, or moiety.

The term “target nucleic acid” or “target” refers to the nucleic acidsequence that is specifically amplified and/or detected using thecompositions, methods and kits of the present teachings (in contrast toa secondary amplification product, which is the result of a spuriousside-reaction, typically due to mis-priming). In certain embodiments, atarget nucleic acid serves as a template in a primer extension reaction.In some embodiments, a target nucleic acid serves as an amplificationtemplate. In some embodiments, a target nucleic acid serves as atemplate strand in a nucleic acid cleavage structure. In certainembodiments, the target nucleic acid comprises DNA and is present ingenomic DNA (gDNA) or mitochondrial DNA (mtDNA). In certain embodiments,the target nucleic acid comprises RNA, for example but not limited to,ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), or anRNA molecule such as a micro RNA (miRNA) precursor, including withoutlimitation, a pri-miRNA, a pre-miRNA, or a pri-miRNA and a pre-miRNA. Insome embodiments, the target nucleic acid comprises a small RNA moleculeincluding without limitation, a miRNA, a sRNA, a stRNA, a snoRNA, orother ncRNA. The target nucleic acid need not constitute the entirety ofa nucleic acid molecule. For example, but not as a limitation, a largenucleic acid, for example, a gDNA fragment, may comprise a multiplicityof different target nucleic acids. Typically, a target nucleic acid hasat least one defined end. In many nucleic acid amplification reactions,the nucleic acid target has two defined ends.

The term “thermostable” when used in reference to an enzyme, refers toan enzyme (such as a polypeptide having nucleic acid polymeraseactivity) that is resistant to inactivation by heat. A “thermostable”enzyme is in contrast to a “thermolabile” polymerase, which can beinactivated by heat treatment. Thermolabile proteins can be inactivatedat physiological temperatures, and can be categorized asmesothermostable (inactivation at about 45° C. to about 65° C.), andthermostable (inactivation at greater than about 65° C.). For example,the activities of the thermolabile T5 and T7 DNA polymerases can betotally inactivated by exposing the enzymes to a temperature of about90° C. for about 30 seconds. A thermostable polymerase activity is moreresistant to heat inactivation than a thermolabile polymerase. However,a thermostable polymerase does not mean to refer to an enzyme that istotally resistant to heat inactivation; thus heat treatment may reducethe polymerase activity to some extent. A thermostable polymerasetypically will also have a higher optimum temperature than thermolabileDNA polymerases.

Working concentration refers to the concentration of a reagent that isat or near the optimal concentration used in a solution to perform aparticular function (such as amplification or digestion of a nucleicacid molecule). The working concentration of a reagent is also describedequivalently as a “1×concentration” or a “1×solution” (if the reagent isin solution) of the reagent. Accordingly, higher concentrations of thereagent may also be described based on the working concentration; forexample, a “2×concentration” or a “2×solution” of a reagent is definedas a concentration or solution that is twice as high as the workingconcentration of the reagent; a “5×concentration” or a “5×solution” isfive times as high as the working concentration, and so on.

As used herein, “nucleic acid polymerase” refers to an enzyme thatcatalyzes the polymerization of nucleotides. Generally, the enzyme willinitiate synthesis at the 3′-end of the primer annealed to a nucleicacid template sequence, and will proceed toward the 5′ end of thetemplate strand. “DNA polymerase” as used herein generally refers to anypolypeptide that can catalyze the 5′-to-3′ extension of a hybridizedprimer by the addition of catalyzes the polymerization ofdeoxynucleotides dideoxyribonucleotides, and/or certain nucleotideanalogs in a template-dependent manner. For example, but not limited to,the sequential addition of deoxyribonucleotides to the 3′-end of aprimer that is annealed to a nucleic acid template during a primerextension reaction. Non-limiting examples of DNA polymerases includeRNA-dependent DNA polymerases, including without limitation, reversetranscriptases, and DNA-dependent DNA polymerases. Known DNA polymerasesinclude, for example, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberget al., 1991, Gene, 108:1), E. coli DNA polymerase I (Lecomte andDoubleday, 1983, Nucleic Acids Res. 11:7505), T7 DNA polymerase(Nordstrom et al., 1981, J. Biol. Chem. 256:3112), Thermus thermophilus(Tth) DNA polymerase (Myers and Gelfand 1991, Biochemistry 30:7661),Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977,Biochim Biophys Acta 475:32), Thermococcus litoralis (Tli) DNApolymerase (also referred to as Vent DNA polymerase, Cariello et al.,1991, Nucleic Acids Res, 19: 4193), 9°Nm DNA polymerase (discontinuedproduct from New England Biolabs), Thermotoga maritima (Tma) DNApolymerase (Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239), Thermusaquaticus (Taq) DNA polymerase (Chien et al., 1976, J. Bacteoriol, 127:1550), Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al., 1997,Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (Patentapplication WO 0132887), and Pyrococcus GB-D (PGB-D) DNA polymerase(Juncosa-Ginesta et al., 1994, Biotechniques, 16:820). The polymeraseactivity of any of the above enzyme can be determined by means wellknown in the art. One unit of DNA polymerase activity, according to thesubject invention, is defined as the amount of enzyme which catalyzesthe incorporation of 10 nmoles of total dNTPs into polymeric form in 30minutes at optimal temperature (e.g., 72° C. for Pfu DNA polymerase). Itis to be appreciated that certain DNA polymerases (for example, but notlimited to certain eubacterial Type A DNA polymerases and Taq DNApolymerase) may further comprise a structure-specific nuclease activity.Reverse transcriptase enzymes suitable for the practice of the presentinvention are also well known in the art and can be derived from anumber of sources. Such enzymes include, but are not limited to, M-MLV,HIV, ASLV and variants and mutants thereof.

As used herein, the terms “amplification”, “nucleic acid amplification”,or “amplifying” refer to the production of multiple copies of a nucleicacid template, or the production of multiple nucleic acid sequencecopies that are complementary to the nucleic acid template. The terms(including the term “polymerizing”) may also refer to extending anucleic acid template (e.g., by polymerization). The amplificationreaction may be a polymerase-mediated extension reaction such as, forexample, a polymerase chain reaction (PCR). However, any of the knownamplification reactions may be suitable for use as described herein. Theterm “amplifying” that typically refers to an “exponential” increase intarget nucleic acid may be used herein to describe both linear andexponential increases in the numbers of a select target sequence ofnucleic acid.

The term “amplification reaction mixture” and/or “master mix” may referto an aqueous solution comprising the various (some or all) reagentsused to amplify a target nucleic acid. Such reactions may also beperformed using solid supports (e.g., an array). The reactions may alsobe performed in single or multiplex format as desired by the user. Thesereactions typically include enzymes, aqueous buffers, salts,amplification primers, target nucleic acid, and nucleosidetriphosphates. Depending upon the context, the mixture can be either acomplete or incomplete amplification reaction mixture. The method usedto amplify the target nucleic acid may be any available to one of skillin the art. Any in vitro means for multiplying the copies of a targetsequence of nucleic acid may be utilized. These include linear,logarithmic, and/or any other amplification method. While thisdisclosure may generally discuss PCR as the nucleic acid amplificationreaction, it is expected that the modified detergents describe hereinshould be effective in other types of nucleic acid amplificationreactions, including both polymerase-mediated amplification reactions(such as helicase-dependent amplification (HDA), recombinase-polymeraseamplification (RPA), and rolling circle amplification (RCA)), as well asligase-mediated amplification reactions (such as ligase detectionreaction (LDR), ligase chain reaction (LCR), and gap-versions of each),and combinations of nucleic acid amplification reactions such as LDR andPCR (see, for example, U.S. Pat. No. 6,797,470). For example, themodified detergents may be used in, for example, variousligation-mediated reactions, where for example ligation probes areemployed as opposed to PCR primers. Additional exemplary methods includepolymerase chain reaction (PCR; see, e.g., U.S. Pat. Nos. 4,683,202;4,683,195; 4,965,188; and/or 5,035,996), isothermal procedures (usingone or more RNA polymerases (see, e.g., PCT Publication No. WO2006/081222), strand displacement (see, e.g., U.S. Pat. No. RE39,007E),partial destruction of primer molecules (see, e.g., PCT Publication No.WO 2006/087574)), ligase chain reaction (LCR) (see, e.g., Wu, et al.,Genomics 4: 560-569 (1990)), and/or Barany, et al. Proc. Natl. Acad.Sci. USA 88:189-193 (1991)), Qβ RNA replicase systems (see, e.g., PCTPublication No. WO 1994/016108), RNA transcription-based systems (e.g.,TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Pat. No.5,854,033; U.S. Patent Application Publication No. 2004/265897; Lizardiet al. Nat. Genet. 19: 225-232 (1998); and/or Banér et al. Nucleic AcidRes., 26: 5073-5078 (1998)), and strand displacement amplification (SDA)(Little, et al. Clin. Chem. 45:777-784 (1999)), among others. Thesesystems, along with the many other systems available to the skilledartisan, may be suitable for use in polymerizing and/or amplifyingtarget nucleic acids for use as described herein.

“Amplification efficiency” may refer to any product that may bequantified to determine copy number (e.g., the term may refer to a PCRamplicon, an LCR ligation product, and/or similar product). Theamplification and/or polymerization efficiency may be determined byvarious methods known in the art, including, but not limited to,determination of calibration dilution curves and slope calculation,determination using qBase software as described in Hellemans et al.,Genome Biology 8:R19 (2007), determination using the delta delta Cq(ΔΔCq) calculation as described by Livak and Schmittgen, Methods 25:402(2001), or by the method as described by Pfaffl, Nucl. Acids Res. 29:e45(2001), all of which are herein incorporated by reference in theirentirety.

In general, PCR thermal cycling includes an initial denaturing step athigh temperature, followed by a repetitive series of temperature cyclesdesigned to allow template denaturation, primer annealing, and extensionof the annealed primers by the polymerase. Generally, the samples areheated initially for about 2 to 10 minutes at a temperature of about 95°C. to denature the double stranded DNA sample. Then, in the beginning ofeach cycle, the samples are denatured for about 10 to 60 seconds,depending on the samples and the type of instrument used. Afterdenaturing, the primers are allowed to anneal to the target DNA at alower temperature, from about 40° C. to about 60° C. for about 20 to 60seconds. Extension of the primers by the polymerase is often carried outat a temperature ranging from about 60° C. to about 72° C. The amount oftime used for extension will depend on the size of the amplicon and thetype of enzymes used for amplification and is readily determined byroutine experimentation. Additionally, the annealing step can becombined with the extension step, resulting in a two step cycling.Thermal cycling may also include additional temperature shifts in PCRassays. The number of cycles used in the assay depends on many factors,including the primers used, the amount of sample DNA present, and thethermal cycling conditions. The number of cycles to be used in any assaymay be readily determined by one skilled in the art using routineexperimentation. Optionally, a final extension step may be added afterthe completion of thermal cycling to ensure synthesis of allamplification products.

PCR with the disclosed compositions can be performed on “standard” PCRinstrumentation, e.g., Applied Biosystems 7900HT, 7500, and 7300standard PCR systems, or on “fast” PCR instrumentation, e.g., AppliedBiosystems StepOne™, StepOne Plus™, 7500 and 7900HT Fast Real-time PCRsystems, or ViiA™ 7 or QuantStudio™ 12K Flex Real-Time PCR systems.

In some embodiments, exemplary thermal cycling conditions for PCRamplification using the compositions and methods disclosed herein are asfollows:

UNG Step (Optional): 50° C. for 2 minutes

Activation: 95° C. for 2 minutes

Denaturation: 95-97° C. for 15 seconds

Annealing/Extension: 60-62° C. for 1 minute (× up to 40 cycles)

In some embodiments, exemplary thermal cycling conditions for PCRamplification using the compositions and methods disclosed herein are asfollows:

UNG Step (Optional): 50° C. for 2 minutes

Activation: 95° C. for 2 minutes

Denaturation: 95-97° C. for 15 seconds

Annealing: 55-60° C. for 15 seconds

Extension: 72° C. for 1 minute (× up to 40 cycles)

In some embodiments, the compositions disclosed herein are used for fastPCR thermal cycling. In one embodiment, fast thermal cycling conditionsfor PCR amplification using the composition and methods disclosed hereinare as follows:

UNG Step (Optional): 50° C. for 2 minutes

Activation: 95° C. for 2 minutes

Denaturation: 95-97° C. for 1-3 seconds

Extension: 60-62° C. for 20-30 seconds (× up to 40 cycles)

In some embodiments, when fast PCR thermal cycling is performed a primerconcentration of about 400 nM is recommended.

In certain embodiments, amplification techniques comprise at least onecycle of amplification, for example, but not limited to, the steps of:denaturing a double-stranded nucleic acid to separate the componentstrands; hybridizing a primer to a target flanking sequence or aprimer-binding site of an amplicon (or complements of either, asappropriate); and synthesizing a strand of nucleotides in atemplate-dependent manner using a DNA polymerase. The cycle may or maynot be repeated. In certain embodiments, a cycle of amplificationcomprises a multiplicity of amplification cycles, for example, but notlimited to 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45cycles or more than 45 cycles of amplification.

In some embodiments, amplifying comprises thermal cycling using aninstrument, such as, but not limited to, a GeneAmp® PCR System 9700,9600, 2700 or 2400 thermocycler, a QuantStudio™ 12K Flex Real-Time PCRSystem, an Applied Biosystems® ViiA™ 7 Real-Time PCR System, an AppliedBiosystems® 7500 Fast Real-Time PCR System, a 7900HT Fast Real-Time PCRSystem, and the like (all available from Life Technologies Corp.,Carlsbad, Calif.). In certain embodiments, single-stranded amplicons aregenerated in an amplification reaction, for example, but not limited toasymmetric PCR or A-PCR.

Devices have been developed that can perform a thermal cycling reactionwith and detection of reaction compositions containing a nucleic acidbinding dye, by emitting a light beam of a specified wavelength, readingthe intensity of the fluorescent signal emitted from the nucleic acidbinding dye molecules associated with double-stranded nucleic acids, anddisplaying the intensity of fluorescence after each cycle. Devicescomprising a thermal cycler, light beam emitter, and a fluorescentsignal detector have been described in, e.g., U.S. Pat. Nos. 5,928,907;6.015,674; and 6,174,670, and include without limitation, the ABI Prism®7700 Sequence Detection System, the GeneAmp® PCR System 9700, 9600, 2700or 2400 thermocycler, the Applied Biosystems® ViiA™ 7 Real-Time PCRSystem, the Applied Biosystems® 7500 Fast Real-Time PCR System, the7900HT Fast Real-Time PCR System, and the like (all available from LifeTechnologies Corp., Carlsbad, Calif.).

In some embodiments, amplification comprises a two-step reactionincluding without limitation, a pre-amplification step wherein a limitednumber of cycles of amplification occur (for example, but not limitedto, 2, 3, 4, or 5 cycles of amplification), then the resulting ampliconis generally diluted and portions of the diluted amplicon are subjectedto additional cycles of amplification in a subsequent amplification step(see, e.g., U.S. Pat. No. 6,605,451 and U.S. Patent ApplicationPublication No. 2004/0175733). In some embodiments, a pre-amplificationstep, a subsequent amplification step, or both, includes a dual hotstart reaction mixture.

In certain embodiments, an amplification reaction comprises multiplexamplification, in which a multiplicity of different target nucleic acidsand/or a multiplicity of different amplification product species aresimultaneously amplified using a multiplicity of different primer sets.In certain embodiments, a multiplex amplification reaction and asingle-plex amplification reaction, including a multiplicity ofsingle-plex or lower-plexy reactions (for example, but not limited to atwo-plex, a three-plex, a four-plex, a five-plex or a six-plex reaction)are performed in parallel.

Exemplary methods for polymerizing and/or amplifying nucleic acidsinclude, for example, polymerase-mediated extension reactions. Forinstance, the polymerase-mediated extension reaction can be thepolymerase chain reaction (PCR). In other embodiments, the nucleic acidamplification reaction is a multiplex reaction. For instance, exemplarymethods for polymerizing and/or amplifying and detecting nucleic acidssuitable for use as described herein are commercially available asTaqMan® (see, e.g., U.S. Pat. Nos. 4,889,818; 5,079,352; 5,210,015;5,436,134; 5,487,972; 5,658,751; 5,210,015; 5,487,972; 5,538,848;5,618,711; 5,677,152; 5,723,591; 5,773,258; 5,789,224; 5,801,155;5,804,375; 5,876,930; 5,994,056; 6,030,787; 6,084,102; 6,127,155;6,171,785; 6,214,979; 6,258,569; 6,814,934; 6,821,727; 7,141,377; and/or7,445,900, all of which are hereby incorporated herein by reference intheir entirety). TaqMan® assays are typically carried out by performingnucleic acid amplification on a target polynucleotide using a nucleicacid polymerase having 5′-to-3′ nuclease activity, a primer capable ofhybridizing to said target polynucleotide, and an oligonucleotide probecapable of hybridizing to said target polynucleotide 3′ relative to saidprimer. The oligonucleotide probe typically includes a detectable label(e.g., a fluorescent reporter molecule) and a quencher molecule capableof quenching the fluorescence of said reporter molecule. Typically, thedetectable label and quencher molecule are part of a single probe. Asamplification proceeds, the polymerase digests the probe to separate thedetectable label from the quencher molecule. The detectable label (e.g.,fluorescence) is monitored during the reaction, where detection of thelabel corresponds to the occurrence of nucleic acid amplification (e.g.,the higher the signal the greater the amount of amplification).Variations of TaqMan® assays (e.g., LNA™ spiked TaqMan® assay) are knownin the art and would be suitable for use in the methods describedherein.

Another exemplary system suitable for use as described herein utilizesdouble-stranded probes in displacement hybridization methods (see, e.g.,Morrison et al. Anal. Biochem., 18:231-244 (1989); and/or Li, et al.Nucleic Acids Res., 30(2,e5) (2002)). In such methods, the probetypically includes two complementary oligonucleotides of differentlengths where one includes a detectable label and the other includes aquencher molecule. When not bound to a target nucleic acid, the quenchersuppresses the signal from the detectable label. The probe becomesdetectable upon displacement hybridization with a target nucleic acid.Multiple probes may be used, each containing different detectablelabels, such that multiple target nucleic acids may be queried in asingle reaction.

Additional exemplary methods for polymerizing and/or amplifying anddetecting target nucleic acids suitable for use as described hereininvolve “molecular beacons”, which are single-stranded hairpin shapedoligonucleotide probes. In the presence of the target sequence, theprobe unfolds, binds and emits a signal (e.g., fluoresces). A molecularbeacon typically includes at least four components: 1) the “loop”, an18-30 nucleotide region which is complementary to the target sequence;2) two 5-7 nucleotide “stems” found on either end of the loop and beingcomplementary to one another; 3) at the 5′ end, a detectable label; and4) at the 3′ end, a quencher moiety that prevents the detectable labelfrom emitting a single when the probe is in the closed loop shape (e.g.,not bound to a target nucleic acid). Thus, in the presence of acomplementary target, the “stem” portion of the beacon separates outresulting in the probe hybridizing to the target. Other types ofmolecular beacons are also known and may be suitable for use in themethods described herein. Molecular beacons may be used in a variety ofassay systems. One such system is nucleic acid sequence-basedamplification (NASBA®), a single step isothermal process forpolymerizing and/or amplifying RNA to double stranded DNA withouttemperature cycling. A NASBA reaction typically requires avianmyeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNApolymerase, RNase H, and two oligonucleotide primers. Afteramplification, the amplified target nucleic acid may be detected using amolecular beacon. Other uses for molecular beacons are known in the artand would be suitable for use in the methods described herein.

The Scorpions™ system is another exemplary assay format that may be usedin the methods described herein. Scorpions™ primers are bi-functionalmolecules in which a primer is covalently linked to the probe, alongwith a detectable label (e.g., a fluorophore) and a non-detectablequencher moiety that quenches the fluorescence of the detectable label.In the presence of a target nucleic acid, the detectable label and thequencher separate which leads to an increase in signal emitted from thedetectable label. Typically, a primer used in the amplification reactionincludes a probe element at the 5′ end along with a “PCR blocker”element (e.g., a hexaethylene glycol (HEG) monomer (Whitcombe, et al.Nat. Biotech. 17: 804-807 (1999)) at the start of the hairpin loop. Theprobe typically includes a self-complementary stem sequence with adetectable label at one end and a quencher at the other. In the initialamplification cycles (e.g., PCR), the primer hybridizes to the targetand extension occurs due to the action of polymerase. The Scorpions™system may be used to examine and identify point mutations usingmultiple probes that may be differently tagged to distinguish betweenthe probes. Using PCR as an example, after one extension cycle iscomplete, the newly synthesized target region will be attached to thesame strand as the probe. Following the second cycle of denaturation andannealing, the probe and the target hybridize. The hairpin sequence thenhybridizes to a part of the newly produced PCR product. This results inthe separation of the detectable label from the quencher and causesemission of the signal. Other uses for such labeled probes are known inthe art and would be suitable for use in the methods described herein.

In some embodiments, the methods are performed before or in conjunctionwith a sequencing reaction. The term “sequencing” is used in a broadsense herein and refers to any technique known in the art that allowsthe order of at least some consecutive nucleotides in at least part of apolynucleotide, for example but not limited to a target nucleic acid oran amplicon, to be identified. Some non-limiting examples of sequencingtechniques include Sanger's dideoxy terminator method and the chemicalcleavage method of Maxam and Gilbert, including variations of thosemethods; sequencing by hybridization; sequencing by synthesis; andrestriction mapping. Some sequencing methods comprise electrophoresis,including capillary electrophoresis and gel electrophoresis; sequencingby hybridization including microarray hybridization; mass spectrometry;single molecule detection; and ion/proton detection. In someembodiments, sequencing comprises direct sequencing, duplex sequencing,cycle sequencing, single base extension sequencing (SBE), solid-phasesequencing, or combinations thereof. In some embodiments, sequencingcomprises detecting the sequencing product using an instrument, forexample but not limited to an ABI Prism® 377 DNA Sequencer, an ABIPrism® 310, 3100, 3100-Avant, 3730 or 3730xl Genetic Analyzer, an ABIPrism® 3700 DNA Analyzer, an Ion PGM™ sequencer, or an Ion Proton™sequencer (all available from Life Technologies Corp., Carlsbad,Calif.), or a mass spectrometer. In some embodiments, sequencingcomprises incorporating a dNTP, including a dATP, a dCTP, a dGTP, adTTP, a dUTP, a dITP, or combinations thereof, and includingdideoxyribonucleotide analogs of dNTPs, into an amplification product.

The nucleic acid polymerases that may be employed in the disclosednucleic acid amplification reactions may be any that function to carryout the desired reaction including, for example, a prokaryotic, fungal,viral, bacteriophage, plant, and/or eukaryotic nucleic acid polymerase.As used herein, the term “DNA polymerase” refers to an enzyme thatsynthesizes a DNA strand de novo using a nucleic acid strand as atemplate. DNA polymerase uses an existing DNA or RNA as the template forDNA synthesis and catalyzes the polymerization of deoxyribonucleotidesalongside the template strand, which it reads. The newly synthesized DNAstrand is complementary to the template strand. DNA polymerase can addfree nucleotides only to the 3′-hydroxyl end of the newly formingstrand. It synthesizes oligonucleotides via transfer of a nucleosidemonophosphate from a deoxyribonucleoside triphosphate (dNTP) to the3′-hydroxyl group of a growing oligonucleotide chain. This results inelongation of the new strand in a 5′-to-3′ direction. Since DNApolymerase can only add a nucleotide onto a pre-existing 3′-OH group, tobegin a DNA synthesis reaction, the DNA polymerase needs a primer towhich it can add the first nucleotide. Suitable primers may compriseoligonucleotides of RNA or DNA, or chimeras thereof (e.g., RNA/DNAchimerical primers). The DNA polymerases may be a naturally occurringDNA polymerases or a variant of natural enzyme having theabove-mentioned activity. For example, it may include a DNA polymerasehaving a strand displacement activity, a DNA polymerase lacking 5′-to-3′exonuclease activity, a DNA polymerase having a reverse transcriptaseactivity, or a DNA polymerase having an endonuclease activity.

Suitable nucleic acid polymerases may also comprise holoenzymes,functional portions of the holoenzymes, chimeric polymerase, or anymodified polymerase that can effectuate the synthesis of a nucleic acidmolecule. Within this disclosure, a DNA polymerase may also include apolymerase, terminal transferase, reverse transcriptase, telomerase,and/or polynucleotide phosphorylase. Non-limiting examples ofpolymerases may include, for example, T7 DNA polymerase, eukaryoticmitochondrial DNA Polymerase γ, prokaryotic DNA polymerase I, II, III,IV, and/or V; eukaryotic polymerase α, β, γ, δ, ε, η, ζ, ι, and/or κ; Ecoli DNA polymerase I; E. coli DNA polymerase III alpha and/or epsilonsubunits; E. coli polymerase IV, E. coli polymerase V; T aquaticus DNApolymerase I; B. stearothermophilus DNA polymerase I; Euryarchaeotapolymerases; terminal deoxynucleotidyl transferase (TdT); S. cerevisiaepolymerase 4; translesion synthesis polymerases; reverse transcriptase;and/or telomerase. Non-limiting examples of suitable thermostable DNApolymerases that may be used include Taq, Tfl, Tf1, Pfu, and Vent™ DNApolymerases, any genetically engineered DNA polymerases, any havingreduced or insignificant 3′-to-5′ exonuclease activity (e.g.,SuperScript™ DNA polymerase), and/or genetically engineered DNApolymerases (e.g., those having the active site mutation F667Y or theequivalent of F667Y (e.g., in Tth), AmpliTaq®FS, ThermoSequenase™ ),AmpliTaq® Gold, Platinum® Taq DNA Polymerase, Therminator I, TherminatorII, Therminator III, Therminator Gamma (all available from New EnglandBiolabs, Beverly, Mass.), and/or any derivatives and fragments thereof.Other nucleic acid polymerases may also be suitable as would beunderstood by one of skill in the art.

In another aspect, the present disclosure provides reaction mixtures forpolymerizing and/or amplifying a nucleic acid sequence of interest(e.g., a target sequence). In some embodiments, the reaction mixture mayfurther comprise a detectable label. The methods may also include one ormore steps for detecting the detectable label to quantitate theamplified nucleic acid. As used herein, the term “detectable label”refers to any of a variety of signaling molecules indicative ofamplification. For example, SYBR® Green and other DNA-binding dyes aredetectable labels. Such detectable labels may comprise or may be, forexample, nucleic acid intercalating agents or non-intercalating agents.As used herein, an intercalating agent is an agent or moiety capable ofnon-covalent insertion between stacked base pairs of a double-strandednucleic acid molecule. A non-intercalating agent is one that does notinsert into the double-stranded nucleic acid molecule. The nucleic acidbinding agent may produce a detectable signal directly or indirectly.The signal may be detectable directly using, for example, fluorescenceand/or absorbance, or indirectly using, for example, any moiety orligand that is detectably affected by proximity to double-strandednucleic acid is suitable such as a substituted label moiety or bindingligand attached to the nucleic acid binding agent. It is typicallynecessary for the nucleic acid binding agent to produce a detectablesignal when bound to a double-stranded nucleic acid that isdistinguishable from the signal produced when that same agent is insolution or bound to a single-stranded nucleic acid. For example,intercalating agents such as ethidium bromide fluoresce more intenselywhen intercalated into double-stranded DNA than when bound tosingle-stranded DNA, RNA, or in solution (see, e.g., U.S. Pat. Nos.5,994,056; 6,171,785; and/or 6,814,934). Similarly, actinomycin Dfluoresces in the red portion of the UV/VIS spectrum when bound tosingle-stranded nucleic acids, and fluoresces in the green portion ofthe UV/VIS spectrum when bound to double-stranded nucleic acids. And inanother example, the photoreactive psoralen4-aminomethyl-4-5′,8-trimethylpsoralen (AMT) has been reported toexhibit decreased absorption at long wavelengths and fluorescence uponintercalation into double-stranded DNA (Johnson et al. Photochem. &Photobiol., 33:785-791 (1981). For example, U.S. Pat. No. 4,257,774describes the direct binding of fluorescent intercalators to DNA (e.g.,ethidium salts, daunomycin, mepacrine and acridine orange,4′,6-diamidino-α-phenylindole). Non-intercalating agents (e.g., minorgroove binders as described herein such as Hoechst 33258, distamycin,netropsin) may also be suitable for use. For example, Hoechst 33258(Searle, et al. Nucl. Acids Res. 18(13):3753-3762 (1990)) exhibitsaltered fluorescence with an increasing amount of target. Minor groovebinders are described in more detail elsewhere herein.

Other DNA binding dyes are available to one of skill in the art and maybe used alone or in combination with other agents and/or components ofan assay system. Exemplary DNA binding dyes may include, for example,acridines (e.g., acridine orange, acriflavine), actinomycin D (Jain, etal. J. Mol. Biol. 68:21 (1972)), anthramycin, BOBO™-1, BOBO™-3,BO-PRO™-1, cbromomycin, DAPI (Kapuseinski, et al. Nucl. Acids Res.6(112): 3519 (1979)), daunomycin, distamycin (e.g., distamycin D), dyesdescribed in U.S. Pat. No. 7,387,887, ellipticine, ethidium salts (e.g.,ethidium bromide), fluorcoumanin, fluorescent intercalators as describedin U.S. Pat. No. 4,257,774, GelStar® (Cambrex Bio Science Rockland Inc.,Rockland, Me.), Hoechst 33258 (Searle and Embrey, Nucl. Acids Res.18:3753-3762 (1990)), Hoechst 33342, homidium, JO-PRO™-1, LIZ dyes,LO-PRO™-1, mepacrine, mithramycin, NED dyes, netropsin,4′,6-diamidino-α-phenylindole, proflavine, POPO™-1, POPO™-3, PO-PRO™-1,propidium iodide, ruthenium polypyridyls, S5, SYBR® Gold, SYBR® Green I(U.S. Pat. Nos. 5,436,134 and 5,658,751), SYBR® Green II, SYTOX® blue,SYTOX® green, SYTO® 43, SYTO® 44, SYTO® 45, SYTOX® Blue, TO-PRO®-1,SYTO® 11, SYTO® 13, SYTO® 15, SYTO® 16, SYTO® 20, SYTO® 23, thiazoleorange (Aldrich Chemical Co., Milwaukee, Wis.), TOTO™-3, YO-PRO®-1, andYOYO®-3 (Molecular Probes, Inc., Eugene, Oreg.), among others. SYBR®Green I (see, e.g., U.S. Pat. Nos. 5,436,134; 5,658,751; and/or6,569,927), for example, has been used to monitor a PCR reactions. OtherDNA binding dyes may also be suitable as would be understood by one ofskill in the art.

For use as described herein, one or more detectable labels and/orquenching agents may be attached to one or more primers and/or probes(e.g., detectable label). The detectable label may emit a signal whenfree or when bound to one of the target nucleic acids. The detectablelabel may also emit a signal when in proximity to another detectablelabel. Detectable labels may also be used with quencher molecules suchthat the signal is only detectable when not in sufficiently closeproximity to the quencher molecule. For instance, in some embodiments,the assay system may cause the detectable label to be liberated from thequenching molecule. Any of several detectable labels may be used tolabel the primers and probes used in the methods described herein. Asmentioned above, in some embodiments the detectable label may beattached to a probe, which may be incorporated into a primer, or mayotherwise bind to amplified target nucleic acid (e.g., a detectablenucleic acid binding agent such as an intercalating or non-intercalatingdye). When using more than one detectable label, each should differ intheir spectral properties such that the labels may be distinguished fromeach other, or such that together the detectable labels emit a signalthat is not emitted by either detectable label alone. Exemplarydetectable labels include, for instance, a fluorescent dye or fluorphore(e.g., a chemical group that can be excited by light to emitfluorescence or phosphorescence), “acceptor dyes” capable of quenching afluorescent signal from a fluorescent donor dye, and the like. Suitabledetectable labels may include, for example, fluorosceins (e.g.,5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Hydroxy Tryptamine (5-HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC;6-carboxy-1,4-dichloro-2′,7′-dichloro-fluorescein (TET);6-carboxy-1,4-dichloro-2′,4′,5′,7′-tetrachlorofluorescein (HEX);6-carboxy-4′,5′-dichloro-2′,7′- dimethoxyfluorescein (JOE); Alexa fluor®fluorophores (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568,594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY® fluorophores(e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568,564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP,FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-XSE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S,AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin,CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes(e.g., calcium crimson, calcium green, calcium orange, calcofluorwhite), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5,5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescentproteins (e.g., green fluorescent protein (e.g., GFP. EGFP), bluefluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyanfluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescentprotein (e.g., YFP, Citrine, Venus, YPet), FRET donor/acceptor pairs(e.g., fluorescein/tetramethylrhodamine, IAEDANS/fluorescein,EDANS/dabcyl, fluorescein/fluorescein, BODIPY® FL/BODIPY® FL,Fluorescein/QSY7 and QSY9), LysoTracker® and LysoSensor™ (e.g.,LysoTracker® Blue DND-22, LysoTracker® Blue-White DPX, LysoTracker®Yellow HCK-123, LysoTracker® Green DND-26, LysoTracker® Red DND-99,LysoSensor™ Blue DND-167, LysoSensor™ Green DND-189, LysoSensor™ GreenDND-153, LysoSensor™ Yellow/Blue DND-160, LysoSensor™ Yellow/Blue 10,000MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines(e.g., 110, 123, B, B 200, BB, BG, B extra,5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G,Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red,Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine),Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA(6-carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), WT),Texas Red, Texas Red-X, VIC and other labels described in, e.g., U.S.Pat. Application Publication No. 2009/0197254 (incorporated herein byreference in its entirety), among others as would be known to those ofskill in the art. Other detectable labels may also be used (see, e.g.,U.S. Pat. Application Publication No. 2009/0197254 (incorporated hereinby reference in its entirety)), as would be known to those of skill inthe art. Any of these systems and detectable labels, as well as manyothers, may be used to detect amplified target nucleic acids.

Some detectable labels may be sequence-based (also referred to herein as“locus-specific detectable label”), for example 5′-nuclease probes. Suchprobes may comprise one or more detectable labels. Various detectablelabels are known in the art, for example (TaqMan® probes describedherein (See also U.S. Pat. No. 5,538,848 (incorporated herein byreference in its entirety)) various stem-loop molecular beacons (See,e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer,Nature Biotechnology 14:303-308 (1996)), stemless or linear beacons(See, e.g., PCT Publication No. WO 99/21881; U.S. Pat. No. 6,485,901),PNA Molecular Beacons™ (See, e.g., U.S. Pat. Nos. 6,355,421 and6,593,091), linear PNA beacons (See, e.g., Kubista et al., SPIE4264:53-58 (2001)), non-FRET probes (See, e.g., U.S. Pat. No.6,150,097), Sunrise®/Amplifluor® probes (U.S. Pat. No. 6,548,250),stem-loop and duplex Scorpions™ probes (Solinas et al., Nucleic AcidsResearch 29:E96 (2001) and U.S. Pat. No. 6,589,743), bulge loop probes(U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250),cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (EpochBiosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleicacid (PNA) light-up probes (Svanvik, et al. Anal Biochem 281:26-35(2001)), self-assembled nanoparticle probes, ferrocene-modified probesdescribed, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al.,Methods 25:463-471 (2001); Whitcombe et al., Nature Biotechnology.17:804-807 (1999); Isacsson et al., Molecular Cell Probes. 14:321-328(2000); Svanvik et al., Anal Biochem. 281:26-35 (2000); Wolffs et al.,Biotechniques 766:769-771 (2001); Tsourkas et al., Nucleic AcidsResearch. 30:4208-4215 (2002); Riccelli et al., Nucleic Acids Research30:4088-4093 (2002); Zhang et al., Acta Biochimica et Biophysica Sinica(Shanghai). 34:329-332 (2002); Maxwell et al., J. Am. Chem. Soc.124:9606-9612 (2002); Broude et al., Trends Biotechnol. 20:249-56(2002); Huang et al., Chem Res. Toxicol. 15:118-126 (2002); and Yu etal., J. Am. Chem. Soc. 14:11155-11161 (2001); QuantiProbes®(www.qiagen.com), HyBeacons® (French, et al. Mol. Cell. Probes15:363-374 (2001)), displacement probes (Li, et al. Nucl. Acids Res.30:e5 (2002)), HybProbes (Cardullo, et al. Proc. Natl. Acad. Sci. USA85:8790-8794 (1988)), MGB Alert (www.nanogen.com), Q-PNA (Fiandaca, etal. Genome Res. 11:609-611 (2001)), Plexor® (www.Promega.com), LUX™primers (Nazarenko, et al. Nucleic Acids Res. 30:e37 (2002)), DzyNAprimers (Todd, et al. Clin. Chem. 46:625-630 (2000)). Detectable labelsmay also comprise non-detectable quencher moieties that quench thefluorescence of the detectable label, inlcuding, for example, black holequenchers (Biosearch), Iowa Black® quenchers (IDT), QSY quencher(Molecular Probes), and Dabsyl and Dabcyl sulfonate/carboxylateQuenchers (Epoch). Detectable labels may also comprise two probes,wherein for example a fluorophore is on one probe, and a quencher is onthe other, wherein hybridization of the two probes together on a targetquenches the signal, or wherein hybridization on a target alters thesignal signature via a change in fluorescence. Exemplary systems mayalso include FRET, salicylate/DTPA ligand systems (see, e.g., Oser etal. Angew. Chem. Int. Engl. 29(10):1167 (1990)), displacementhybridization, homologous probes, and/or assays described in EuropeanPatent No. EP 070685 and/or U.S. Pat. No. 6,238,927. Detectable labelscan also comprise sulfonate derivatives of fluorescein dyes with SO₃instead of the carboxylate group, phosphoramidite forms of fluorescein,phosphoramidite forms of Cy5 (available for example from Amersham). Allreferences cited above are hereby incorporated herein by reference intheir entirety.

The compositions and methods described herein may be useful fordetecting and/or quantifying a variety of target nucleic acids from atest sample. A target nucleic acid is any nucleic acid for which anassay system is designed to identify or detect as present (or not),and/or quantify in a test sample. Such nucleic acids may include, forexample, those of infectious agents (e.g., virus, bacteria, parasite,and the like), a disease process such as cancer, diabetes, or the like,or to measure an immune response. Exemplary “test samples” includevarious types of samples, such as biological samples. Exemplarybiological samples include, for instance, a bodily fluid (e.g., blood,saliva, spinal fluid), a tissue sample, a food (e.g., meat) or beverage(e.g., milk) product, or the like. Expressed nucleic acids may include,for example, genes for which expression (or lack thereof) is associatedwith medical conditions such as infectious disease (e.g., bacterial,viral, fungal, protozoal infections) or cancer. The methods describedherein may also be used to detect contaminants (e.g., bacteria, virus,fungus, and/or protozoan) in pharmaceutical, food, or beverage products.The methods described herein may be also be used to detect rare allelesin the presence of wild type alleles (e.g., one mutant allele in thepresence of 10⁶ -10⁹ wild type alleles). The methods are useful to, forexample, detect minimal residual disease (e.g., rare remaining cancercells during remission, especially mutations in the p53 gene or othertumor suppressor genes previously identified within the tumors), and/ormeasure mutation load (e.g., the frequency of specific somatic mutationspresent in normal tissues, such as blood or urine).

Kits for performing the methods described herein are also provided. Asused herein, the term “kit” refers to a packaged set of relatedcomponents, typically one or more compounds or compositions. The kit maycomprise a pair of oligonucleotides for polymerizing and/or amplifyingat least one target nucleic acid from a sample, one or more detergents,a nucleic acid polymerase), a dual hot start reaction mixture, and/orcorresponding one or more probes labeled with a detectable label. Thekit may also include samples containing pre-defined target nucleic acidsto be used in control reactions. The kit may also optionally includestock solutions, buffers, enzymes, detectable labels or reagentsrequired for detection, tubes, membranes, and the like that may be usedto complete the amplification reaction. In some embodiments, multipleprimer sets are included. In one embodiment, the kit may include one ormore of, for example, a buffer (e.g., Tris), one or more salts (e.g.,KCl), glycerol, dNTPs (dA, dT, dG, dC, dU), recombinant BSA (bovineserum albumin), a dye (e.g., ROX passive reference dye), one or moredetergents, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP),and/or gelatin (e.g., fish or bovine source). Other embodiments ofparticular systems and kits are also contemplated which would beunderstood by one of skill in the art.

EXAMPLES

Commercially available master mixes with different hot start mechanismswere combined and tested to demonstrate the effectiveness of a dual hotstart reaction mixture. The experimental setup is outlined in Table 1:

TABLE 1 Experimental Setup Reaction Conditions Set 1 Fast SYBR ® GreenMaster Mix (alone) Fast SYBR ® Green Master Mix + Platinum ® SYBR ®Green qPCR SuperMix (1:1 ratio) Platinum ® SYBR ® Green qPCR SuperMix(alone) Set 2 Fast SYBR ® Green Master Mix (alone) Fast SYBR ® GreenMaster Mix + Platinum ® Taq Antibody Set 3 Power SYBR ® Green PCR MasterMix (alone) Power SYBR ® Green PCR Master Mix + Platinum ® Taq Antibody

All reactions were performed with 2 ng Universal Human Reference (UHR)(Agilent Technologies, Santa Clara, Calif.) cDNA and 200 nM primers (24targets tested) in a final volume of 10 μl. Four replicates wereperformed for each reaction. Amplification was performed on a ViiA™ 7Real-Time PCR System (Life Technologies Corp., Carlsbad, Calif.) afterincubation at room temperature for either 0 hours or 24 hours (T0 andT24, respectively). After incubation at room temperature, amplificationwas carried out according to the manufacturer's instructions. Briefly,thermal cycling conditions were set as follows:

Standard Cycling Mode (Primer T_(m) ≥ 60° C.) Step Temperature DurationCycles UDG Activation 50° C. 2 min Hold AmpliTaq ®DNA 95° C. 2 min HoldPolymerase, UP Activation Denature 95° C. 15 sec 40 Anneal/Extend 60° C.1 min Standard Cycling Mode (Primer T_(m) ≥ 60° C.) Step TemperatureDuration Cycles UDG Activation 50° C. 2 min Hold AmpliTaq ®DNA 95° C. 2min Hold Polymerase, UP Activation Denature 95° C. 15 sec 40 Anneal55-60° C.*  15 sec Extend 72° C. 1 min

In some instances, Power SYBR® Green qPCR SuperMix or Fast SYBR® GreenMaster Mix was incubated overnight (16-20 hours) with Platinum® TaqAntibody. All components tested are commercially available and werepurchased from Life Technologies Corporation.

In FIGS. 1A-1D, the target nucleic acid was amplified with 24 primersets using either a single or a dual hot start reaction mixture. Thesingle hot start reaction mixtures used either Fast SYBR® Green MasterMix alone (“square” line) or Platinum® SYBR® Green qPCR SuperMix alone(“cross” line). The dual hot start reaction mixture used a 1:1combination of Fast SYBR® Green Master Mix and Platinum® SYBR® GreenqPCR SuperMix (“triangle” line). All three reaction sets were incubatedat room temperature for either 0 hours (“T0”) or 24 hours (“T24”) beforeamplification. In many of the assays, the combination of the twodifferent hot start mechanisms resulted in a decrease in the formationof non-specific products (see, FIGS. 1A (T0, cDNA), 1B (T0, non-specificproduct (“NTC”)), 1C (T24, cDNA), and 1D (T24, NTC)).

In FIGS. 2A-1 through 2L-2 , individual amplicons were selected andanalyzed by melt curve analysis. FIG. 2A-1 through 2C-2 demonstratesthat the dual hot start reaction mixture gives a 4-fold reduction in theformation of non-specific products when compared to the single hot startreaction mixture (Fast SYBR® Green Master Mix) and a 2-fold reductionwhen compared to the other single hot start reaction mixture (Platinum®SYBR® Green qPCR SuperMix) after 0 hours at room temperature. FIG. 2D-1through 2F-2 demonstrates that the dual hot start reaction mixture givesa 2-fold reduction in the formation of non-specific products whencompared to each of the single hot start reaction mixtures after 24 hourpre-incubation at room temperature. FIGS. 2G-1 through 2I-2 and 2J-1through 2L-2 show that the dual hot start reaction mixture gives atleast at 50% reduction in non-specific products at 0 hourspre-incubation at room temperature (FIG. 2G-1 through 2I-2) and a 100%reduction in non-specific products after 24 hours pre-incubation at roomtemperature (FIG. 2J-1 through 2L-2). FIG. 2M compares the mean Ct andthe ΔCt analysis of amplification reactions of the single and dual hotstart reaction mixtures effect on reduction of non-specific productformation. FIG. 2N shows a comparison of the melt curve analyses of thesingle and dual hot start reaction mixtures demonstrating that the dualhot start reaction mixture significantly reduces the formation ofnon-specific products at both 0 and 24 hours pre-incubation at roomtemperature (as shown by a reduction in the NTC ΔRn signal).

In FIGS. 3A-3D, the target nucleic acid was amplified with 24 primersets using either a single or a dual hot start reaction mixture. Thesingle hot start reaction mixtures used either Fast SYBR® Green MasterMix alone (“diamond” line) or in combination with Platinum® Taq Antibody(“cross” line). Both reaction sets were incubated at room temperaturefor either 0 hours (“T0”) or 24 hours (“T24”) before amplification. Inmany of the assays, the combination of the two different hot startmechanisms resulted in a decrease in the formation of non-specificproducts (see, FIGS. 3A (T0, cDNA), 3B (T0, non-specific product(“NTC”)), 3C (T24, cDNA), and 3D (T24, NTC)).

In FIGS. 4A-1 through 4F-2, individual amplicons were selected andanalyzed by melt curve analysis. FIG. 4A-1 through 4B-2 demonstratesthat the Fast SYBR® Green Master Mix single hot start reaction mixtureproduces non-specific products, especially after a 24 hourpre-incubation at room temperature. FIG. 4C-1 through 4E demonstratesthat the dual hot start reaction mixture results in a 75% reduction innon-specific product formation at T0 and a 100% reduction innon-specific product formation at T24. FIG. 4F demonstrates that thedual hot start reaction mixture reduces the formation of non-specificproducts at both T0 and T24 and that the dual hot start reaction mixturereduces the formation of non-specific products by 20-25% (depending onthe threshold value used).

In FIGS. 5A-5D, the target nucleic acid was amplified with 24 primersets using either a single or a dual hot start reaction mixture. Thesingle hot start reaction mixtures used either Power SYBR® Green PCRMaster Mix alone (“diamond” line) or in combination with Platinum® TaqAntibody (“cross” line). Both reaction sets were incubated at roomtemperature for either 0 hours (“T0”) or 24 hours (“T24”) beforeamplification. In many of the assays, the combination of the twodifferent hot start mechanisms resulted in a decrease in the formationof non-specific products (see, FIGS. 5A (T0, cDNA), 5B (T0, non-specificproduct (“NTC”)), 5C (T24, cDNA), and 5D (T24, NTC)).

In FIGS. 6A-1 through 6E-2, individual amplicons were selected andanalyzed by melt curve analysis. FIG. 6A-1 through 6B-2 demonstratesthat the Power SYBR® Green PCR Master Mix single hot start reactionmixture produces non-specific products, especially after a 24 hourpre-incubation at room temperature. FIG. 6C-1 through 6D-2 demonstratesthat the dual hot start reaction mixture results in a 50% reduction innon-specific product formation at 24 hours. FIG. 6E demonstrates thatthe dual hot start reaction mixture reduces the formation ofnon-specific products at both T0 and T24 (as shown by a drop in Ctvalues) and that the dual hot start reaction mixture reduces theformation of non-specific products by 3-fold at T0 and by 10% at T24.

We claim:
 1. A composition comprising: a) a nucleic acid polymerase; andb) a dual hot start reaction mixture.
 2. The composition of claim 1,wherein the nucleic acid polymerase is a DNA-dependent DNA polymerase oran RNA-dependent DNA polymerase.
 3. The composition of claim 1, whereinthe nucleic acid polymerase is thermostable.
 4. The composition of claim1, wherein the nucleic acid polymerase is selected from the groupconsisting of Taq DNA polymerase, Tfl DNA polymerase, Tfi DNApolymerase, Pfu DNA polymerase, and Vent™ DNA polymerase.
 5. Thecomposition of claim 1, wherein the dual hot start reaction mixturecomprises at least two different hot start mechanisms.
 6. Thecomposition of claim 5, wherein the at least two different hot startmechanisms are selected from the group consisting of antibodies orcombinations of antibodies that block DNA polymerase activity at lowertemperatures, oligonucleotides that block DNA polymerase activity atlower temperatures, reversible chemical modifications of the DNApolymerase that dissociate at elevated temperatures, amino acidmodifications of the DNA polymerase that provide reduced activity atlower temperatures, fusion proteins that include hyperstable DNA bindingdomains and topoisomerase, temperature dependent ligands that inhibitthe DNA polymerase, single stranded binding proteins that sequesterprimers at lower temperatures, modified primers, or modified dNTPs.
 7. Acomposition comprising: a) a thermostable nucleic acid polymerase; andb) a dual hot start reaction mixture that substantially inhibits thepolymerase activity of the polymerase at a temperature less than about40° C. and wherein the dual hot start reaction mixture does notsubstantially inhibit the polymerase activity of the polymerase at atemperature greater than about 40° C.
 8. The composition of claim 7,wherein the nucleic acid polymerase is a DNA-dependent DNA polymerase oran RNA-dependent DNA polymerase.
 9. The composition of claim 7, whereinthe nucleic acid polymerase is selected from the group consisting of TaqDNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, Pfu DNApolymerase, and Vent™ DNA polymerase.
 10. The composition of claim 7,wherein the dual hot start reaction mixture comprises at least twodifferent hot start mechanisms.
 11. The composition of claim 10, whereinthe at least two different hot start mechanisms are selected from thegroup consisting of antibodies or combinations of antibodies that blockDNA polymerase activity at lower temperatures, oligonucleotides thatblock DNA polymerase activity at lower temperatures, reversible chemicalmodifications of the DNA polymerase that dissociate at elevatedtemperatures, amino acid modifications of the DNA polymerase thatprovide reduced activity at lower temperatures, fusion proteins thatinclude hyperstable DNA binding domains and topoisomerase, temperaturedependent ligands that inhibit the DNA polymerase, single strandedbinding proteins that sequester primers at lower temperatures, modifiedprimers, or modified dNTPs.
 12. A method for amplifying a target nucleicacid comprising: a) contacting the target nucleic acid with a nucleicacid polymerase, a dual hot start reaction mixture, at least one primerand at least one dNTP at a first temperature, thereby forming a reactioncomposition; b) heating the reaction composition to a secondtemperature; and c) amplifying the target nucleic acid in the reactioncomposition.
 13. The method of claim 12, wherein the nucleic acidpolymerase is thermostable.
 14. The method of claim 12, wherein thenucleic acid polymerase is a DNA-dependent DNA polymerase or anRNA-dependent DNA polymerase.
 15. The method of claim 12, wherein thenucleic acid polymerase is selected from the group consisting of Taq DNApolymerase, Tfl DNA polymerase, Tfi DNA polymerase, Pfu DNA polymerase,and Vent™ DNA polymerase.
 16. The method of claim 12, wherein the dualhot start reaction mixture comprises at least two different hot startmechanisms.
 17. The method of claim 16, wherein the at least twodifferent hot start mechanisms are selected from the group consisting ofantibodies or combinations of antibodies that block DNA polymeraseactivity at lower temperatures, oligonucleotides that block DNApolymerase activity at lower temperatures, reversible chemicalmodifications of the DNA polymerase that dissociate at elevatedtemperatures, amino acid modifications of the DNA polymerase thatprovide reduced activity at lower temperatures, fusion proteins thatinclude hyperstable DNA binding domains and topoisomerase, temperaturedependent ligands that inhibit the DNA polymerase, single strandedbinding proteins that sequester primers at lower temperatures, modifiedprimers, or modified dNTPs.
 18. A kit comprising: a) a nucleic acidpolymerase; and b) a dual hot start reaction mixture.
 19. The kit ofclaim 18, further comprising at least one primer, dNTPs, and a nucleicacid binding dye.
 20. The kit of claim 18, wherein the dual hot startreaction mixture comprises at least two different hot start mechanisms.